Structure (v.15, #12)
Observation of Decreased Radiation Damage at Higher Dose Rates in Room Temperature Protein Crystallography
by Robert J. Southworth-Davies; Melissa A. Medina; Ian Carmichael; Elspeth F. Garman (pp. 1531-1541).
Radiation damage can be a problem when utilizing ionizing X-radiation in macromolecular crystallography. The dose dependence of radiation damage to eight lysozyme crystals at room temperature (292 K) was investigated in order to provide an accurate comparison with cryotemperature (100 K) results and to allow researchers to calculate expected maximum room-temperature-crystal lifetimes prior to data collection. Results of intensity-loss analysis unexpectedly showed that the dose tolerated by a crystal is dependent on the dose rate according to a positive linear relationship (99% correlation coefficient); a 60% increase in dose rate gave a 4-fold increase in crystal lifetime over the range studied. Alternative metrics of damage were also assessed from room temperature data. In the dose-rate range tested (6 Gy s−1 to 10 Gy s−1), data collection at 100 K appears to offer a 26–113 times increase in the lifetime of the crystal.
Feast/Famine Regulation by Transcription Factor FL11 for the Survival of the Hyperthermophilic Archaeon Pyrococcus OT3
by Katsushi Yokoyama; Sanae A. Ishijima; Hideaki Koike; Chitose Kurihara; Ai Shimowasa; Mamiko Kabasawa; Tsuyoshi Kawashima; Masashi Suzuki (pp. 1542-1554).
Transcriptional repressor FL11 from the hyperthermophilic archaeon, Pyrococcus OT3, was crystallized in its dimer form in complex with a DNA duplex, TGAAAWWWTTTCA. Chemical contacting of FL11 to the terminal 5 bps, and DNA bending by propeller twisting atWWW confirmed specificity of the interaction. Dimer-binding sites were identified in promoters of ∼200 transcription units coding, for example, H+-ATPase and NAD(P)H dehydrogenase. In the presence of lysine, four FL11 dimers were shown to assemble into an octamer, thereby covering the fl11 promoter. In the “feast” mode, when P. OT3 grows on amino acids, the FL11 octamer will terminate transcription of fl11, as was shown in vitro, thereby derepressing transcription of many metabolic genes. In the “famine” mode in the absence of lysine, ∼6000 FL11 dimers present per cell will arrest growth. This regulation resembles global regulation by Escherichia coli leucine-responsive regulatory protein, and hints at a prototype of transcription regulations now highly diverged.
Keywords: PROTEINS; DNA
Quaternary Structure Change as a Mechanism for the Regulation of Thymidine Kinase 1-Like Enzymes
by Dario Segura-Peña; Joseph Lichter; Manuela Trani; Manfred Konrad; Arnon Lavie; Stefan Lutz (pp. 1555-1566).
The human cytosolic thymidine kinase (TK) and structurally related TKs in prokaryotes play a crucial role in the synthesis and regulation of the cellular thymidine triphosphate pool. We report the crystal structures of the TK homotetramer from Thermotoga maritima in four different states: its apo-form, a binary complex with thymidine, as well as the ternary structures with the two substrates (thymidine/AppNHp) and the reaction products (TMP/ADP). In combination with fluorescence spectroscopy and mutagenesis experiments, our results demonstrate that ATP binding is linked to a substantial reorganization of the enzyme quaternary structure, leading to a transition from a closed, inactive conformation to an open, catalytic state. We hypothesize that these structural changes are relevant to enzyme function in situ as part of the catalytic cycle and serve an important role in regulating enzyme activity by amplifying the effects of feedback inhibitor binding.
Modeling Backbone Flexibility Improves Protein Stability Estimation
by Shuangye Yin; Feng Ding; Nikolay V. Dokholyan (pp. 1567-1576).
In designing mutagenesis experiments, it is often crucial to know how certain mutations will affect the structure and thermodynamic stability of the protein. Here, we present a methodology, Eris, to efficiently and accurately compute the stability changes of proteins upon mutations using our protein-modeling suite, Medusa. We evaluate the stability changes upon mutations for 595 mutants from five structurally unrelated proteins, and find significant correlations between the predicted and experimental results. For cases when the high-resolution protein structure is not available, we find that better predictions are obtained by backbone structure prerelaxation. The advantage of our approach is that it is based on physical descriptions of atomic interactions, and does not rely on parameter training with available experimental protein stability data. Unlike other methods, Eris also models the backbone flexibility, thereby allowing for determination of the mutation-induced backbone conformational changes. Eris is freely available via the web server athttp://eris.dokhlab.org.
A CTP-Dependent Archaeal Riboflavin Kinase Forms a Bridge in the Evolution of Cradle-Loop Barrels
by Moritz Ammelburg; Marcus D. Hartmann; Sergej Djuranovic; Vikram Alva; Kristin K. Koretke; Jörg Martin; Guido Sauer; Vincent Truffault; Kornelius Zeth; Andrei N. Lupas; Murray Coles (pp. 1577-1590).
Proteins of the cradle-loop barrel metafold are formed by duplication of a conserved βαβ-element, suggesting a common evolutionary origin from an ancestral group of nucleic acid-binding proteins. The basal fold within this metafold, the RIFT barrel, is also found in a wide range of enzymes, whose homologous relationship with the nucleic acid-binding group is unclear. We have characterized a protein family that is intermediate in sequence and structure between the basal group of cradle-loop barrels and one family of RIFT-barrel enzymes, the riboflavin kinases. We report the structure, substrate-binding mode, and catalytic activity for one of these proteins, Methanocaldococcus jannaschii Mj0056, which is an archaeal riboflavin kinase. Mj0056 is unusual in utilizing CTP rather than ATP as the donor nucleotide, and sequence conservation in the relevant residues suggests that this is a general feature of archaeal riboflavin kinases.
Phosphorylation-Induced Conformational Switching of CPI-17 Produces a Potent Myosin Phosphatase Inhibitor
by Masumi Eto; Toshio Kitazawa; Fumiko Matsuzawa; Sei-ichi Aikawa; Jason A. Kirkbride; Noriyoshi Isozumi; Yumi Nishimura; David L. Brautigan; Shin-ya Ohki (pp. 1591-1602).
Phosphorylation of endogenous inhibitor proteins for type-1 Ser/Thr phosphatase (PP1) provides a mechanism for reciprocal coordination of kinase and phosphatase activities. A myosin phosphatase inhibitor protein CPI-17 is phosphorylated at Thr38 through G-protein-mediated signals, resulting in a >1000-fold increase in inhibitory potency. We show here the solution NMR structure of phospho-T38-CPI-17 with rmsd of 0.36 ± 0.06 Å for the backbone secondary structure, which reveals how phosphorylation triggers a conformational change and exposes an inhibitory surface. This active conformation is stabilized by the formation of a hydrophobic core of intercalated side chains, which is not formed in a phospho-mimetic D38 form of CPI-17. Thus, the profound increase in potency of CPI-17 arises from phosphorylation, conformational change, and hydrophobic stabilization of a rigid structure that poses the phosphorylated residue on the protein surface and restricts its hydrolysis by myosin phosphatase. Our results provide structural insights into transduction of kinase signals by PP1 inhibitor proteins.
Keywords: PROTEINS; SIGNALING
The NMDA Receptor NR1 C1 Region Bound to Calmodulin: Structural Insights into Functional Differences between Homologous Domains
by Zeynep Akyol Ataman; Lokesh Gakhar; Brenda R. Sorensen; Johannes W. Hell; Madeline A. Shea (pp. 1603-1617).
Calmodulin (CaM) regulates tetrameric N-methyl-D-aspartate receptors (NMDARs) by binding tightly to the C0 and C1 regions of its NR1 subunit. A crystal structure (2HQW; 1.96 Å) of calcium-saturated CaM bound to NR1C1 (peptide spanning 875–898) showed that NR1 S890, whose phosphorylation regulates membrane localization, was solvent protected, whereas the endoplasmic reticulum retention motif was solvent exposed. NR1 F880 filled the CaM C-domain pocket, whereas T886 was closest to the N-domain pocket. This 1–7 pattern was most similar to that in the CaM-MARCKS complex. Comparison of CaM-ligand wrap-around conformations identified a core tetrad of CaM C-domain residues (FLMMC) that contacted all ligands consistently. An identical tetrad of N-domain residues (FLMMN) made variable sets of contacts with ligands. This CaM-NR1C1 structure provides a foundation for designing mutants to test the role of CaM in NR1 trafficking as well as insights into how the homologous CaM domains have different roles in molecular recognition.
Keywords: PROTEINS; CELLBIO; MOLNEURO
Transformation Efficiency of RasQ61 Mutants Linked to Structural Features of the Switch Regions in the Presence of Raf
by Greg Buhrman; Glenna Wink; Carla Mattos (pp. 1618-1629).
Transformation efficiencies of Ras mutants at residue 61 range over three orders of magnitude, but the in vitro GTPase activity decreases 10-fold for all mutants. We show that Raf impairs the GTPase activity of RasQ61L, suggesting that the Ras/Raf complex differentially modulates transformation. Our crystal structures show that, in transforming mutants, switch II takes part in a network of hydrophobic interactions burying the nucleotide and precatalytic water molecule. Our results suggest that Y32 and a water molecule bridging it to the γ-phosphate in the wild-type structure play a role in GTP hydrolysis in lieu of the Arg finger in the absence of GAP. The bridging water molecule is absent in the transforming mutants, contributing to the burying of the nucleotide. We propose a mechanism for intrinsic hydrolysis in Raf-bound Ras and elucidate structural features in the Q61 mutants that correlate with their potency to transform cells.
Keywords: PROTEINS; SIGNALING
Combining Efficient Conformational Sampling with a Deformable Elastic Network Model Facilitates Structure Refinement at Low Resolution
by Gunnar F. Schröder; Axel T. Brunger; Michael Levitt (pp. 1630-1641).
Structural studies of large proteins and protein assemblies are a difficult and pressing challenge in molecular biology. Experiments often yield only low-resolution or sparse data that are not sufficient to fully determine atomistic structures. We have developed a general geometry-based algorithm that efficiently samples conformational space under constraints imposed by low-resolution density maps obtained from electron microscopy or X-ray crystallography experiments. A deformable elastic network (DEN) is used to restrain the sampling to prior knowledge of an approximate structure. The DEN restraints dramatically reduce over-fitting, especially at low resolution. Crossvalidation is used to optimally weight the structural information and experimental data. Our algorithm is robust even for noise-added density maps and has a large radius of convergence for our test case. The DEN restraints can also be used to enhance reciprocal space simulated annealing refinement.
Structural Basis of the Initial Binding of tRNAIle Lysidine Synthetase TilS with ATP and L-Lysine
by Mitsuo Kuratani; Yuka Yoshikawa; Yoshitaka Bessho; Kyoko Higashijima; Takeshi Ishii; Rie Shibata; Seizo Takahashi; Katsuhide Yutani; Shigeyuki Yokoyama (pp. 1642-1653).
In the bacterial genetic-code system, the codon AUA is decoded as isoleucine by tRNAIle2 with the lysidine residue at the wobble position. Lysidine is derived from cytidine, with ATP and L-lysine, by tRNAIle lysidine synthetase (TilS), which is an N-type ATP pyrophosphatase. In this study, we determined the crystal structure of Aquifex aeolicus TilS, complexed with ATP, Mg2+, and L-lysine, at 2.5 Å resolution. The presence of the TilS-specific subdomain causes the active site to have two separate gateways, a large hole and a narrow tunnel on the opposite side. ATP is bound inside the hole, and L-lysine is bound at the entrance of the tunnel. The conserved Asp36 in the PP-motif coordinates Mg2+. In these initial binding modes, the ATP, Mg2+, and L-lysine are held far apart from each other, but they seem to be brought together for the reaction upon cytidine binding, with putative structural changes of the complex.
Open-State Conformation of the KcsA K+ Channel: Monte Carlo Normal Mode Following Simulations
by Gennady V. Miloshevsky; Peter C. Jordan (pp. 1654-1662).
Potassium channels fluctuate between closed and open states. The detailed mechanism of the conformational changes opening the intracellular pore in the K+ channel from Streptomyces lividans (KcsA) is unknown. Applying Monte Carlo normal mode following, we find that gating involves rotation and unwinding of the TM2 bundle, lateral movement of the TM2 helices away from the channel axis, and disappearance of the TM2 bundle. The open-state conformation of KcsA exhibits a very wide inner vestibule, with a radius ∼5–7 Å and inner helices bent at the A98–G99 hinge. Computed conformational changes demonstrate that spin labeling and X-ray experiments illuminate different stages in gating: transition begins with clockwise rotation of the TM2 helices ending at a final state with the TM2 bend hinged near residues A98–G99. The concordance between the computational and experimental results provides atomic-level insights into the structural rearrangements of the channel's inner pore.
Keywords: PROTEINS; CELLBIO
Crystal Structure of AcrB in Complex with a Single Transmembrane Subunit Reveals Another Twist
by Susanna Törnroth-Horsefield; Pontus Gourdon; Rob Horsefield; Lars Brive; Natsuko Yamamoto; Hirotada Mori; Arjan Snijder; Richard Neutze (pp. 1663-1673).
Bacterial drug resistance is a serious concern for human health. Multidrug efflux pumps export a broad variety of substrates out of the cell and thereby convey resistance to the host. In Escherichia coli, the AcrB:AcrA:TolC efflux complex forms a principal transporter for which structures of the individual component proteins have been determined in isolation. Here, we present the X-ray structure of AcrB in complex with a single transmembrane protein, assigned by mass spectrometry as YajC. A specific rotation of the periplasmic porter domain of AcrB is also revealed, consistent with the hypothesized “twist-to-open” mechanism for TolC activation. Growth experiments with yajc-deleted E. coli reveal a modest increase in the organism's susceptibility to β-lactam antibiotics, but this effect could not conclusively be attributed to the loss of interactions between YajC and AcrB.
Structural Basis for the PufX-Mediated Dimerization of Bacterial Photosynthetic Core Complexes
by Johan Busselez; Magali Cottevieille; Philippe Cuniasse; Francesca Gubellini; Nicolas Boisset; Daniel Lévy (pp. 1674-1683).
In Rhodobacter (Rba.) sphaeroides, the subunit PufX is involved in the dimeric organization of the core complex. Here, we report the 3D reconstruction at 12 Å by cryoelectron microscopy of the core complex of Rba. veldkampii, a complex of ∼300 kDa without symmetry. The core complex is monomeric and constituted by a light-harvesting complex 1 (LH1) ring surrounding a uniquely oriented reaction center (RC). The LH1 consists of 15 resolved α/β heterodimers and is interrupted. Within the opening, PufX polypeptide is assigned at a position facing the QB site of the RC. This core complex is different from a dissociated dimer of the core complex of Rba. sphaeroides revealing that PufX in Rba. veldkampii is unable to dimerize. The absence in PufX of Rba. veldkampii of a G31XXXG35 dimerization motif highlights the transmembrane interactions between PufX subunits involved in the dimerization of the core complexes of Rhodobacter species.